1. Field of the Invention
This invention is directed to novel synthetic processes for the preparation of the trisaccharide glycoside .alpha.Gal(1.fwdarw.4).beta.Gal(1.fwdarw.4)Glc-OR. Specifically, this invention is directed to a multi-step synthesis of this trisaccharide aglycon wherein the attachment of the aglycon is conducted after formation of the blocked .beta.Gal(1.fwdarw.4)Glc disaccharide which, surprisingly, significantly enhances the overall synthetic yield of the process.
2. References
The following publications, patents and patent applications are cited in this application as superscript numbers:
.sup.1 Armstrong, et al., U.S. patent application Ser. No. 08/126,645 (allowed) for "Diagnosis and Treatment of Bacterial Dysentery", filed Sep. 27, 1993. PA1 .sup.2 Ratcliffe, et al., U.S. Pat. No. 5,079,353, for "SIALIC ACID GLYCOSIDES, ANTIGENS, IMMUNOADSORBENTS, AND METHODS FOR THEIR PREPARATION" issued Jan. 7, 1992. PA1 .sup.3 Ekborg, et al., "Synthesis of Three Disaccharides for the Preparation of Immunogens Bearing Immunodeterminants Known to Occur on Glycoproteins", Carbohydr. Res., 110:55-67 (1982). PA1 .sup.4 Dahmen, et al., "2-Bromoethyl Glycosides: Applications in the Synthesis of Spacer-Arm Glycosides Carbohydr. Res.", 118:292-301. (1983) PA1 .sup.5 Rana, et al., "Synthesis of Phenyl 2-Acetamido-2-Deoxy-3-O-.alpha.-L-Fucopyranosyl-.beta.-D-Glucopyranoside and Related Compounds", Carbohydr. Res., 91:149-157 (1981). PA1 .sup.6 Amvam-Zollo, et al., "Type XIV Polysaccharide: Synthesis of a Repeating Branched Tetrasaccharide with Dioxa-Type Spacer-Arms" Carbohydr. Res., 150:199-212 (1986). PA1 .sup.7 Paulsen, et al., "Synthese Von Oligosaccharid-Determinanten Mit Amid-Spacer Vom Typ Des T-Antigens*", Carbohydr. Res., 104:195-219 (1984). PA1 .sup.8 Chernyak, et al., "New Type of Carbohydrate-Containing Synthetic Antigen: Synthesis of Carbohydrate-Containing Polyacrylamide Copolymers Having the Specificity of 0:3 and 0:4 Factors of Salmonella", Carbohydr. Res., 128:269-282 (1984). PA1 .sup.9 Fernadez-Santana, et al., "Glycosides of Monoallyl Diethylene Glycol. A new Type of Spacer Group for Synthetic Oligosaccharides", J. Carbohydr. Chem., 8:531-537 (1989). PA1 .sup.10 Lee, et al., "Synthesis of 3-(2-aminoethylthio)propyl Glycosides", Carbohydr. Res., 37:193 et seq., (1974). PA1 .sup.11 Lemieux, et al., "Properties of a "Synthetic" Antigen Related to the Human Blood-Group Lewis a", J. Am. Chem. Soc., 97:4076-4083 (1975) PA1 .sup.12 Pinto, et al., "Preparation of Glycoconjugates for Use as Artificial Antigens: A Simplified Procedure", Carbohydr. Chem., 124:313-318 (1983) PA1 .sup.13 Rafter, et al., U.S. patent application Ser. No. 08/669,004, for "Treatment of Bacterial Dysentery" filed Jun. 21, 1996
All of the above publications, patent applications and patents are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
3. State of the Art
Trisaccharide glycosides, such as the .alpha.Gal(1.fwdarw.4).beta.Gal(1.fwdarw.4)Glc-OR trisaccharide, have been disclosed by Armstrong, et al..sup.1 as binding to Shiga Like Toxins (SLTs) expressed by pathogenic E. coli which can populate the intestinal tract of humans and cause diarrhea. In extreme cases, the pathology of this disease mediated by such toxins progresses to kidney involvement in the form of hemolytic uremic syndrome (HUS) which has a relatively high mortality rate. Accordingly, pharmaceutical compositions comprising such trisaccharides have been proposed for treatment of diarrhea mediated by SLTs..sup.13
Notwithstanding the beneficial properties of such trisaccharides, current synthetic processes for these compounds involve a multi-step process with overall low yields. This, in turn, has hampered the commercial development of these compounds.
Specifically, the complete chemical synthesis of oligosaccharide glycosides is a difficult task involving the generation of differentially protected or blocked hydroxyl groups on at least some of the hydroxyl groups of each of the saccharide units so as to provide a means to selectively remove one or more of the blocking groups thereby permitting the necessary reactions to be conducted on the unblocked hydroxyl group(s) as required to generate the desired compound.sup.2. Additionally, the numerous reaction procedures required in blocking and deblocking different hydroxyl groups necessitate a multi-step chemical synthetic procedure and the generation of crystalline intermediates during the synthetic procedure is certainly desirable in providing a facile means to purify the intermediates other than by chromatography or other equivalent means. In this regard, chromatography on intermediates and products achieved by large scale synthesis of trisaccharide glycosides is recognized as a time consuming process which can require the use of expensive equipment and is generally disadvantageous to an efficient large scale overall synthesis of the desired trisaccharide glycoside.
Contrarily, the use of glycosyltransferases to effect overall synthesis of the desired trisaccharide glycoside can be hindered by the lack of ready availability of the required glycosyltransferase, the difficulty in effecting large scale enzymatic reactions, the difficulty in coupling the desired saccharide to the nucleotide base required for coupling, etc.